Skip to main content
Download PDF

Tracing meristem cell lineages during plant development and regeneration by the all-in-one CRE/LOX system

Plant Methods volume 21, Article number: 36 (2025) Cite this article

Abstract

The root apical meristem (RAM) and the shoot apical meristem (SAM), which contain stem cells and their lineages, are the centers for underground and aboveground organ formation in vascular plants. Accurately tracing stem cell lineage is important to understand how the RAM and SAM are formed and how they give rise to new organs. Here, we introduce the all-in-one CRE/LOX system, which is assembled in a single plasmid and can be used to efficiently trace cell lineage during de novo formation of meristems and cell differentiation within meristems in Arabidopsis thaliana. Using the CRE/LOX-GUS system driven by the WUSCHEL-RELATED HOMEOBOX5 (WOX5) promoter (i.e., WOX5pro-CRE/LOX-GUS), we show that cell division of the quiescent center (QC) within the stem cell niche of the RAM might replenish initial/stem cells in plants grown on mannitol-containing medium or in soil. The results obtained using WOX5pro-CRE/LOX-GUS also show that a group of shoot progenitor cells acts together to initiate the SAM during de novo shoot regeneration from callus in tissue culture. We also demonstrate the use of the CRE/LOX-RUBY system for real-time in vivo tracing of cell lineages in live organs. Overall, this paper not only introduces the all-in-one CRE/LOX tool, but also reveals the cell lineages during meristem development and regeneration.

Introduction

Plant meristems, which contain a group of undetermined and actively dividing cells, are the center of organ formation [1]. In vascular plants, underground and aboveground organs are initiated from the root apical meristem (RAM) and the shoot apical meristem (SAM), respectively. How the meristem is formed and how the meristem gives rise to new organs are key questions in plant development.

During post embryonic development of Arabidopsis thaliana, all the root tissues are differentiated from the RAM. The RAM is composed of the stem cell niche and transit-amplifying cells. The stem cell niche contains the quiescent center (QC) and the initial cells (i.e., stem cells) that surround the QC. Division of the initial/stem cells gives rise to transit-amplifying cells, and the daughter cells derived from transit-amplifying cell division differentiate to produce root tissues [1].

Arabidopsis can create root branches by de novo formation of the RAM [2, 3]. In acropetal lateral root (hereafter referred to as lateral root) formation, several xylem-pole pericycle cells function together as the lateral root founder cells that divide to initiate the lateral root primordium. The continuous division of the lateral root primordium forms the lateral root meristem [4]. In adventitious root formation from detached leaves (i.e., de novo root regeneration), a vascular adult stem cell near the wound serves as the adventitious root founder cell. This founder cell divides and initiates the adventitious root primordium, which gives rise to the adventitious root meristem via continuous cell division [5]. The WUSCHEL (WUS)-RELATED HOMEOBOX (WOX) family transcription factor gene WOX11 is expressed in the adventitious root founder cell but not in lateral root founder cells, and its expression level decreases during the division of the adventitious root founder cell to form the adventitious root primordium [6,7,8]. The WOX11 expression in the adventitious root founder cell is directly activated by the auxin signaling pathway through the auxin response elements (AuxREs) on the WOX11 promoter [6]. In root meristems, WOX11 is expressed in proto-xylem cells [6, 7]. WOX5 expression is activated during the transition from both lateral and adventitious root founder cells to the root primordium, and then its expression is enriched in the QC in root meristems [9,10,11,12]. WOX11 and the AP2-like transcription factors PLETHORA3/5/7 can directly bind to the WOX5 promoter to active the WOX5 expression during the fate transition from adventitious root founder cell to adventitious root primordium [11, 13].

In two-step tissue culture of Arabidopsis hypocotyls, the hypocotyl explants are first cultured on callus-inducing medium (CIM) with a high auxin level. This induces the explants to form pluripotent callus in preparation for organ regeneration. Then, the callus is moved to shoot-inducing medium (SIM) with a high cytokinin level to induce shoot regeneration [14, 15]. On CIM, a vascular adult stem cell within the hypocotyl explant serves as the callus founder cell, which expresses WOX11 activated by auxin [6, 16, 17]. Then, the callus founder cell divides to form the callus primordium that expresses WOX5 [6, 16,17,18]. Continuous cell division of the callus primordium gives rise to callus with three cell layers (i, e. the outer, middle, and inner cell layers), and WOX5 is enriched in the middle cell layer [16, 17]. After the callus is moved to SIM, shoot progenitor cells form in the middle cell layer by expressing WUS [16, 19,20,21,22,23,24]. Then, shoot progenitor cells can enter into two fate directions: the productive shoot progenitor cells continuously divide, express SAM-related genes, and finally form the SAM, while the pseudo shoot progenitor cells lose the ability to form a SAM [24]. Although the SAM can be traced back to a single cell in the explant before culture on CIM [25], it is still unclear whether a single shoot progenitor cell or a group of shoot progenitor cells is responsible for SAM formation on SIM.

The developmental and regenerative processes described above provide efficient platforms for studying meristem cell lineages. The CRE/LOX system has been widely used to trace cell lineages in animals [26,27,28] and plants [16, 28,29,30,31,32]. In our previous studies, we used a CRE/LOX system comprising two constructs, i.e., CRE in one construct and LOX in another construct. The constructs were separately transformed into Arabidopsis, and the cell lineage was traced by crossing the two transformed Arabidopsis lines harboring each construct [29]. In this study, we report the all-in-one CRE/LOX system that combines CRE and LOX in one construct and utilizes the RUBY reporter [33] for real-time and in vivo cell lineage tracing in live organs.

Materials and methods

All-in-one CRE/LOX system construction

The all-in-one CRE/LOX system was constructed based on the two separate plasmids reported previously [29]. Firstly, the 35S promoter in pCAMBIA1300-LOX2272-eGFP-NOSter-LOX2272-GUS-NOSter was replaced with the Arabidopsis UBIQUITIN 10 (UBQ10) promoter (UBQ10pro) [34] to create the UBQ10pro-LOX2272-eGFP-NOSter-LOX2272-GUS-NOSter fragment. Secondly, this fragment was inserted after pBI101-intCRE-GR-HSPter to create pBI101-CRE/LOX-GUS. The pBI101-intCRE-GR-HSPter plasmid contained a intCRE-GLUCOCORTICOID RECEPTOR (GR) sequence by inserting the first intron of castor bean CATALASE1 (CAT1) intron [35] into CRE (intCRE) as well as the HEAT SHOCK PROTEIN 18.2 (HSP18.2) terminator (HSPter) [36]. The β-GLUCURONIDASE (GUS) gene in pBI101-CRE/LOX-GUS was replaced with RUBY [33] to create pBI101-CRE/LOX-RUBY. The 5-kb WOX5 promoter was cloned into pBI101-CRE/LOX-GUS to create pBI101-WOX5pro-CRE/LOX-GUS and the 5-kb WOX11 promoter was cloned into pBI101-CRE/LOX-RUBY to create pBI101-WOX11pro-CRE/LOX-RUBY.

The constructs were introduced into the wild-type Arabidopsis Columbia-0 (Col-0) via Agrobacterium tumefaciens-mediated transformation. Two from a total of twelve transgenic pBI101-WOX5pro-CRE/LOX-GUS lines and five from a total of sixteen transgenic pBI101-WOX11pro-CRE/LOX-RUBY lines showed normal DEX-induced GUS and RUBY expression, respectively. The homozygous plants from those normal DEX-induced GUS or RUBY expression lines all showed reporter activities. Therefore, these lines were used in this study.

The forward and reverse primers used for cloning are listed below: 5′-GAATTCTCTAGAGAGTCAGTAATAAACGG-3′ and 5′-ACTAGTAAGCTTCTGTTAATCAGAAAAAC-3′ for the UBQ10 promoter; 5′-ACTGATTTCGACCAGGTAAATTTCTAGTTTTTCTC-3′ and 5′-CATGAGTGAACGAACCTGTAACTATCATCATCATC-3′ for the CAT1 intron; 5′-TCTAGAATATGAAGATGAAGATGAAA-3′ and 5′-ACTAGTCTTATCTTTAATCATATTCC-3′ for the HSP18.2 terminator; 5′-ACAATTACCAACAACAACAAAC-3′ and 5′-CGACTCACTATAGGGTCTAGTCCCGATCTAGTAACATAGATGAC-3′ for RUBY; 5′-ACGCGTCGACTCAGAGACCAAATTATTTTGG-3′ and 5′-GGTACCGTTCAGATGTAAAGTCCTCAACTG-3′ for the WOX5 promoter; and 5′-TTCGGAGAGCTCGGAGGTACCTGAGCTCATCTAACTGTTACG-3′ and 5′-CATAGATCTGCTAGCGTCGACTGCTTTGAAGAATATTGATAT-3′ for the WOX11 promoter.

Plant materials, plant growth, regeneration conditions, and GUS staining assay

The WOX5pro:GUS line was described previously [37].

For adventitious root regeneration from detached leaves, Arabidopsis seeds were germinated and grown on ½ Murashige & Skoog basal (MS) medium with 1% w/v sucrose at 22 °C under a 16-hour light/8-hour dark photoperiod for 12 days. The first pair of rosette leaves from 12-day-old seedlings was cut at the petiole, and the detached leaves were cultured on B5 medium without sucrose at 22 °C under 24-hour light conditions.

For shoot regeneration in tissue culture, Arabidopsis seeds were germinated and vertically grown on ½ MS medium (1.2% w/v agar) in the light for 1 day and then in the dark for 8 days. Elongated hypocotyls (about 1 cm) were cut and incubated on CIM [MS medium, 2% w/v sucrose, pH 5.8, 0.8% w/v agar, 11 μM 2,4-dichlorophenoxyacetic acid (2,4-D), 0.2 μM kinetin] under 24-hour light conditions for 5 days and 30 min to induce callus formation. Callus was transferred to SIM [MS medium, 2% w/v sucrose, pH 5.7, 0.8% w/v agar, 2 μM 6-(dimethylallylamino) purine (2iP), 0.9 μM indole acetic acid (IAA)] and cultured under 24-hour light conditions for 7 days.

For analyses of lateral roots and RAMs in plants growing on medium, Arabidopsis seeds were germinated and grown vertically on ½ MS medium containing 1% w/v sucrose at 22 °C under a 16-hour light/8-hour dark photoperiod.

For analyses of RAMs in plants grown in soil, Arabidopsis seeds were germinated and grown in soil at 22 °C under a 16-hour light/8-hour dark photoperiod.

Details of the GUS staining method have been reported previously [37].

Accession numbers

Sequence data from this article can be found in the Arabidopsis Genome Initiative under the following accession numbers: WOX5 (AT3G11260), WOX11 (AT3G03660), HSP18.2 (AT5G59720), and UBQ10 (AT4G05320).

Results

All-in-one pBI101-CRE/LOX-GUS system

In our previous study, we used a CRE/LOX system based on two constructs [29]. Here, we combined all those elements into one construct based on the pBI101 plasmid (Fig. 1A and B). The all-in-one construct pBI101-CRE/LOX-GUS consisted of two parts. First, the intCRE gene (CRE gene with an intron insertion to avoid accumulation of its encoded protein in bacteria) was fused with a dexamethasone (DEX)-induced nuclear localization domain GR sequence [38]. This intCRE-GR fused gene was terminated with the HSPter [36]. Tissue-specific promoters can be inserted into the multiple cloning site to drive the expression of intCRE-GR. Second, the fused fragment LOX2272-eGFP-NOSter-LOX2272 was inserted between the UBQ10pro [34] and the GUS reporter gene to block the expression of GUS driven by UBQ10pro.

Fig. 1
figure 1

The pBI101-CRE/LOX-GUS system. A. Schematic of the all-in-one CRE/LOX-GUS system to trace cell lineage. B. The plasmid vector pBI101-CRE/LOX-GUS. MCS, multiple cloning site

Full size image

To use this all-in-one pBI101-CRE/LOX-GUS system, a tissue-specific promoter must be cloned into the multiple cloning site. After the construct is transformed into plants, the intCRE-GR gene is transcribed under the control of the tissue-specific promoter. To trace cell lineage, DEX is used to allow the CRE-GR protein into the nuclei, then CRE-GR implements the recombination of the two LOX2272 sequences to form a single LOX2272 sequence, resulting in the activation of GUS expression. In this way, cells with the GUS signal are confirmed as descendants of the cell(s) in which the initial LOX2272 recombination event occurred (Fig. 1A).

To test this system, we cloned the WOX5 promoter into pBI101-CRE/LOX-GUS to form pBI101-WOX5pro-CRE/LOX-GUS and transformed this construct into the wild-type Arabidopsis Col-0 background. The pBI101-WOX5pro-CRE/LOX-GUS transgenic line was grown vertically on ½ MS medium for 7 days without DEX treatment, and then moved to DEX-containing medium and grown for another 3 days. The results show that some lateral roots were clearly labeled with the GUS signal (Fig. 2A). When developing lateral roots were observed under a differential interference contrast (DIC) microscope, GUS signals were observed at all stages of lateral root primordia and the lateral root meristem (Fig. 2B–E). This result confirms that WOX5 expression is activated in the lateral root primordium, and all the cells of the lateral root are descendants of the WOX5-expressing lateral root primordium [12, 29].

Fig. 2
figure 2

Tracing lateral and adventitious root cell lineages using the pBI101-WOX5pro-CRE/LOX-GUS system. A–E. GUS staining of the pBI101-WOX5pro-CRE/LOX-GUS transgenic line, showing positive GUS signals in lateral roots (A), lateral root primordia (B–D), and lateral root meristem (E). Seedlings were germinated and grown vertically on ½ MS DEX-free medium (no DEX) for 7 days (d), then cultured on ½ MS medium containing 10 μM DEX (+ DEX) for 3 days. F. Diagram of adventitious root formation from detached Arabidopsis leaves in (G). G. GUS staining of the pBI101-WOX5pro-CRE/LOX-GUS transgenic line, showing positive GUS signals in adventitious roots growing from detached leaves. Seedlings were cultured for 12 days on DEX-free ½ MS medium, and then two leaves (first leaf pair) were detached from the same plant. One was cultured on B5 medium with 10 μM DEX (+ DEX) for 7 days, and the other was cultured on B5 medium without DEX (no DEX) for 7 days as the control. Two independent lines were analyzed and showed the same results. Scale bars, 1 mm in (A, G) and 50 μm in (B–E)

Full size image

We also tested the pBI101-WOX5pro-CRE/LOX-GUS system during adventitious root regeneration from detached Arabidopsis leaves (Fig. 2F). The detached leaves of the pBI101-WOX5pro-CRE/LOX-GUS transgenic line were cultured on B5 medium with DEX for 7 days, and all the regenerated adventitious roots were labeled with GUS signals (Fig. 2G). In the control (no DEX), there were no GUS signals in the adventitious roots (Fig. 2G). These observations confirm the findings of previous studies, i.e., that WOX5 is activated in the adventitious root primordium, and that all the cells of the adventitious root are descendants of the WOX5-expressing adventitious root primordium [11, 29].

Overall, the all-in-one pBI101-CRE/LOX-GUS is a reliable system showing the same results compared with the previous reported two-construct CRE/LOX-GUS system [29] in lateral and adventitious rooting.

Cell lineages in the RAM cultured on medium or in soil

Next, we analyzed the cell lineage in the RAM. The pBI101-WOX5pro-CRE/LOX-GUS transgenic plants were cultured vertically on DEX-free ½ MS medium without mannitol for 3 days and then moved to DEX-containing ½ MS medium without (mock) or with mannitol to impose osmotic stress for another 2 days (Fig. 3A). The primary roots in the plants treated with mannitol were shorter than those in the plants with the mock treatment (Fig. 3B–D).

Fig. 3
figure 3

Cell lineage in the stem cell niche of RAM under osmotic stress. A. Diagram of mannitol treatment to impose stress. pBI101-WOX5pro-CRE/LOX-GUS transgenic seedlings were grown vertically on DEX-free ½ MS medium (no DEX) for 3 days (d). Then, seedlings were transferred to ½ MS medium containing 10 μM DEX (+ DEX) containing 400 mM mannitol or without mannitol (0 mM mannitol as the mock) and grown for a further 2 days (B–H). B–D. Phenotype (B, C) and statistical (D) analyses of the pBI101-WOX5pro-CRE/LOX-GUS transgenic seedlings grown in mock or osmotic stress conditions. Data are mean values ± SD (n = 47 in mock and n = 46 in mannitol treatment). ** P < 0.01 in two-tailed Student’s t tests, and individual values (black dots) and mean values (bars) are shown (D). E–G. GUS staining in the pBI101-WOX5pro-CRE/LOX-GUS transgenic line cultured with 0 mM (E, mock) and 400 mM (F, G) mannitol. Note that GUS signals were restricted to QC cells in 0 mM mannitol treatment (mock) (E) but were present in QC cells, vascular initial cells, and cortex and endodermis initial cells in mannitol treatments (F, G). A total of 9 roots were observed in mock conditions, and all of them showed GUS signals in the QC cells; and a total of 19 roots were observed under 400 mM mannitol treatment, and 9 of them showed GUS signals in initial cells. H. GUS staining in the WOX5pro:GUS transgenic line cultured with 400 mM mannitol. A total of 20 roots were observed, and all of them showed GUS signals in the QC cells. Two independent lines were analyzed and showed the same results. Scale bars, 5 mm in (B, C) and 10 μm in (E–H)

Full size image

Under mock conditions, GUS signals were observed in the QC, indicating that the WOX5-marked QC cells did not divide to replenish initial/stem cells (Fig. 3E). Interestingly, in the mannitol treatment, the GUS signals in the pBI101-WOX5pro-CRE/LOX-GUS line were present in the vascular initial cells and cortex and endodermis initial cells in addition to the QC cells (Fig. 3F and G). However, in the mannitol treatment, the GUS signals in the WOX5pro:GUS line were still observed in the QC (Fig. 3H). Therefore, these data indicated that the WOX5-marked QC cells might divide to replenish the initial/stem cells.

We also cultured the pBI101-WOX5pro-CRE/LOX-GUS and WOX5pro:GUS lines in soil without DEX for 3 days, and then with DEX for another 4 days (Fig. 4A and B). The GUS signals in the WOX5pro:GUS line were enriched in the QC cells (Fig. 4E); but those in the pBI101-WOX5pro-CRE/LOX-GUS line were present in cortex and endodermis initial cells, the columella stem cells, and the columella cells in addition to the QC cells (Fig. 4C and D). Thus, the WOX5-marked QC cells divided to replenish initial/stem cells when the plants were grown in soil.

Fig. 4
figure 4

Cell lineage in the stem cell niche of RAM in soil. A, B. Diagram (A) and pictures (B) of seedlings grown in soil. Seeds were germinated and seedlings were grown in DEX-free soil (no DEX) for 3 days (d), then watered every 12 h with an aqueous solution of 100 μM DEX (+ DEX) for 4 days (C–E). C, D. GUS staining of the root of the 7-day-old pBI101-WOX5pro-CRE/LOX-GUS transgenic line grown in soil with DEX treatment. Note that GUS signals were present in cortex and endodermis initial cells (C), columella stem cells (D), and columella cells (D) in soil. A total of 19 roots were observed, and 8 of them showed GUS signals in initial/stem cells or columella cells in addition to the QC cells. E. GUS staining of the root of the 5-day-old WOX5pro:GUS transgenic line grown in soil. A total of 19 roots were observed, and all of them showed GUS signals in the QC cells. Two independent lines were analyzed and showed the same results. Scale bars, 1 mm in (B) and 50 μm in (C–E)

Full size image

Cell lineages in SAM regeneration during tissue culture

In the two-step tissue culture of Arabidopsis hypocotyls, WOX5 expression is enriched in the middle cell layer of callus, where shoot progenitor cells may arise [16,17,18]. To analyze the cell lineage during SAM formation from callus in tissue culture, we established two different culture conditions. In the first condition, we cultured the hypocotyls of the pBI101-WOX5pro-CRE/LOX-GUS line on CIM containing DEX for 5 days and 30 min (constant DEX treatment) to form callus, and then the callus was cultured on SIM without DEX for 7 days (Fig. 5A). In the second condition, we cultured the hypocotyls of the pBI101-WOX5pro-CRE/LOX-GUS line on CIM without DEX for 5 days and then with DEX for 30 min (transient DEX treatment) to form callus, and then the callus was cultured on SIM without DEX for 7 days (Fig. 5C).

Fig. 5
figure 5

Cell lineage during SAM formation in tissue culture. A, B. Diagram (A) and GUS staining result (B) of SAM regeneration in tissue culture with constant DEX treatment on CIM. Hypocotyl explants from pBI101-WOX5pro-CRE/LOX-GUS transgenic line were cultured on CIM with 10 μM DEX (+ DEX) for 5 days and 30 min to form callus, which was then moved to SIM without DEX (no DEX) for 7 days. C, D. Diagram (C) and GUS staining result (D) of SAM regeneration in tissue culture with transient DEX treatment on CIM. Hypocotyl explants from pBI101-WOX5pro-CRE/LOX-GUS transgenic line were cultured on CIM for 5 days without DEX, moved to CIM with 10 μM DEX for 30 min, then callus was moved to SIM without DEX (no DEX) and cultured for 7 days. Blue asterisks indicate cells with LOX2272 recombination event, showing positive GUS staining signals (A, C). Two independent lines were analyzed and showed the same results. Scale bars, 100 μm in (B, D)

Full size image

In the first condition (constant DEX treatment), the CRE protein had sufficient time for LOX2272 recombination in almost all cells in the middle cell layer. Consequently, GUS signals were observed in almost all cells in the regenerated SAM (Fig. 5A and B), consistent with previous observations using the two-construct CRE/LOX system [16].

In the second condition with transient DEX treatment on CIM, the CRE protein had limited time for LOX2272 recombination in some of the cells in the middle cell layer, resulting in a mosaic of positive and negative GUS signals in the regenerated SAM (Fig. 5C and D). This result indicates that the SAM arises not from a single shoot progenitor cell, but from a group of shoot progenitor cells on SIM.

All-in-one pBI101-CRE/LOX-RUBY system

To trace the cell lineage in live organs in a real-time and in vivo manner, we constructed the pBI101-CRE/LOX-RUBY system (Fig. 6A and B) by replacing the GUS gene in pBI101-CRE/LOX-GUS with RUBY, which encodes the enzymes that convert tyrosine to red betalain [33]. Betalain is visible to the naked eye without further staining treatments.

Fig. 6
figure 6

The pBI101-CRE/LOX-RUBY system. A. Schematic of the all-in-one CRE/LOX-RUBY system to trace cell lineage in vivo. B. Schematic of plasmid vector pBI101-CRE/LOX-RUBY. C, D. Images of the pBI101-WOX11pro-CRE/LOX-RUBY transgenic line, showing positive red betalain signal in adventitious roots developed from detached leaves. Seedlings were germinated on DEX-free ½ MS medium, grown for 12 days, and first-pair leaves were detached and cultured on B5 medium with 10 μM DEX treatment (+ DEX) for 7 days. Two independent lines were analyzed and showed the same results. Scale bars, 1 mm in (C, D)

Full size image

To test this system, we cloned the WOX11 promoter into pBI101-CRE/LOX-RUBY, generating pBI101-WOX11pro-CRE/LOX-RUBY. The pBI101-WOX11pro-CRE/LOX-RUBY construct was transformed into wild-type Arabidopsis Col-0. The red betalain signal was clearly observed in adventitious roots regenerating from detached leaves of the pBI101-WOX11pro-CRE/LOX-RUBY line (Fig. 6C and D), supporting the previous finding that adventitious roots originate from WOX11-marked adventitious root founder cells [6].

Discussion

In this study, we describe the all-in-one CRE/LOX system that is able to trace cell lineage based on GUS signals or RUBY-mediated red betalain signals.

Using this system, we found that stress conditions might have effects on the cell lineage or cell identity in the RAM. The pBI101-WOX5pro-CRE/LOX-GUS lines showed positive GUS signals in the initial/stem cells in addition to the QC when the roots were under osmotic stress conditions or grown in soil. It is possible that the QC cells within the RAM divide and replenish initial/stem cells when the root is grown under stress conditions or in soil. Our previous study also indicated that the QC contributes to the root cap cell lineage and the vascular cell lineage when the plant was vertically grown on ½ MS medium for a long time [29]. It was proposed that the QC might serve as a cell reservoir for reconstituting damaged cells within the meristem, and QC could exhibit active cell division in response to damage in the RAM [39,40,41,42,43]. It will be important to comprehensively analyze the RAM development mechanism and cell lineages in the RAM in roots growing under stress conditions and in soil in the future.

Previous studies have proposed that the SAM might beformed from multiple cells in plant materials cultured on SIM in tissue culture [24, 44]. Our data support this idea, because we observed that the SAM formed from a group of shoot progenitor cells, rather than a single shoot progenitor cell. It is important to note that during Agrobacterium-mediated gene transformation or gene editing, the regenerated plant might be a mosaic if the Agrobacterium is added after materials have been cultured on CIM or SIM for some time. It is not clear why only groups of shoot progenitor cells are able to establish the fate of productive shoot progenitor cells, which can further proliferate into a SAM. One possibility is that the formation of heterogeneous gene expression patterns within the shoot progenitor cell group is essential for the fate establishment of the productive shoot progenitor cells, leading to the SAM formation [24]. In further studies, it will be interesting to study the mechanism by which shoot progenitor cells collaboratively give rise to the productive shoot progenitor cells in plant tissue culture on SIM.

Data availability

No datasets were generated or analysed during the current study.

References

  1. Scheres B. Stem-cell niches: nursery rhymes across kingdoms. Nat Rev Mol Cell Biol. 2007;8:345–54. Available from: http://www.nature.com/articles/nrm2164

  2. Birnbaum KD. How many ways are there to make a root? Curr Opin Plant Biol. 2016;34:61–7. Available from: https://linkinghub.elsevier.com/retrieve/pii/S1369526616301571

  3. Motte H, Vanneste S, Beeckman T. Molecular and environmental regulation of root development. Annu Rev Plant Biol. 2019;70:465–88. Available from: https://www.annualreviews.org/doi/https://doiorg.publicaciones.saludcastillayleon.es/10.1146/annurev-arplant-050718-100423

  4. Banda J, Bellande K, von Wangenheim D, Goh T, Guyomarc’h S, Laplaze L et al. Lateral root formation in Arabidopsis: a well-ordered LRexit. Trends Plant Sci. 2019;24:826–39. Available from: https://doiorg.publicaciones.saludcastillayleon.es/10.1016/j.tplants.2019.06.015

  5. Xu L. De novo root regeneration from leaf explants: wounding, auxin, and cell fate transition. Curr Opin Plant Biol. 2018;41:39–45. Available from: https://doiorg.publicaciones.saludcastillayleon.es/10.1016/j.pbi.2017.08.004

  6. Liu J, Sheng L, Xu Y, Li J, Yang Z, Huang H et al. WOX11 and 12 are involved in the first-step cell fate transition during de novo root organogenesis in Arabidopsis. Plant Cell. 2014;26:1081–93. Available from: http://www.plantcell.org/cgi/doi/https://doiorg.publicaciones.saludcastillayleon.es/10.1105/tpc.114.122887

  7. Sheng L, Hu X, Du Y, Zhang G, Huang H, Scheres B et al. Non-canonical WOX11-mediated root branching contributes to plasticity in Arabidopsis root system architecture. Development. 2017;144:3126–33. Available from: http://dev.biologists.org/lookup/doi/https://doiorg.publicaciones.saludcastillayleon.es/10.1242/dev.152132

  8. Zhang T, Ge Y, Cai G, Pan X, Xu L. WOX-ARF modules initiate different types of roots. Cell Rep. 2023;42:112966. Available from: https://doiorg.publicaciones.saludcastillayleon.es/10.1016/j.celrep.2023.112966

  9. Sarkar AK, Luijten M, Miyashima S, Lenhard M, Hashimoto T, Nakajima K et al. Conserved factors regulate signalling in Arabidopsis thaliana shoot and root stem cell organizers. Nature. 2007;446:811–4. Available from: http://www.nature.com/articles/nature05703

  10. Pi L, Aichinger E, van der Graaff E, Llavata-Peris CI, Weijers D, Hennig L et al. Organizer-derived WOX5 signal maintains root columella stem cells through chromatin-mediated repression of CDF4 expression. Dev Cell. 2015;33:576–88. Available from: https://doiorg.publicaciones.saludcastillayleon.es/10.1016/j.devcel.2015.04.024

  11. Hu X, Xu L. Transcription factors WOX11/12 directly activate WOX5/7 to promote root primordia initiation and organogenesis. Plant Physiol. 2016;172:2363–73. Available from: http://www.plantphysiol.org/lookup/doi/https://doiorg.publicaciones.saludcastillayleon.es/10.1104/pp.16.01067

  12. Du Y, Scheres B. PLETHORA transcription factors orchestrate de novo organ patterning during Arabidopsis lateral root outgrowth. Proc Natl Acad Sci. 2017;114:11709–14. Available from: http://www.pnas.org/lookup/doi/https://doiorg.publicaciones.saludcastillayleon.es/10.1073/pnas.1714410114

  13. Liu W, Zhang Y, Fang X, Tran S, Zhai N, Yang Z et al. Transcriptional landscapes of de novo root regeneration from detached Arabidopsis leaves revealed by time-lapse and single-cell RNA sequencing analyses. Plant Commun. 2022;3:100306. Available from: https://linkinghub.elsevier.com/retrieve/pii/S2590346222000530

  14. Chen C, Hu Y, Ikeuchi M, Jiao Y, Prasad K, Su YH et al. Plant regeneration in the new era: from molecular mechanisms to biotechnology applications. Sci China Life Sci. 2024;67:1338–67. Available from: https://link.springer.com/https://doiorg.publicaciones.saludcastillayleon.es/10.1007/s11427-024-2581-2

  15. Ikeuchi M, Favero DS, Sakamoto Y, Iwase A, Coleman D, Rymen B et al. Molecular mechanisms of plant regeneration. Annu Rev Plant Biol. 2019;70:377–406. Available from: https://www.annualreviews.org/doi/https://doiorg.publicaciones.saludcastillayleon.es/10.1146/annurev-arplant-050718-100434

  16. Zhai N, Xu L. Pluripotency acquisition in the middle cell layer of callus is required for organ regeneration. Nat Plants. 2021;7:1453–60. Available from: https://www.nature.com/articles/s41477-021-01015-8

  17. Zhai N, Pan X, Zeng M, Xu L. Developmental trajectory of pluripotent stem cell establishment in Arabidopsis callus guided by a quiescent center-related gene network. Development. 2023;150:dev200879. Available from: https://journals.biologists.com/dev/article/doi/10.1242/dev.200879/286991/Developmental-trajectory-of-pluripotent-stem-cell

  18. Sugimoto K, Jiao Y, Meyerowitz EM. Arabidopsis regeneration from multiple tissues occurs via a root development pathway. Dev Cell. 2010;18:463–71. Available from: https://linkinghub.elsevier.com/retrieve/pii/S1534580710000924

  19. Gordon SP, Heisler MG, Reddy GV, Ohno C, Das P, Meyerowitz EM. Pattern formation during de novo assembly of the Arabidopsis shoot meristem. Development. 2007;134:3539–48. Available from: http://dev.biologists.org/cgi/doi/https://doiorg.publicaciones.saludcastillayleon.es/10.1242/dev.010298

  20. Zhang T-Q, Lian H, Zhou C-M, Xu L, Jiao Y, Wang J-W. A two-step model for de novo activation of WUSCHEL during plant shoot regeneration. Plant Cell. 2017;29:1073–87. Available from: http://www.ncbi.nlm.nih.gov/pubmed/28389585

  21. Dai X, Liu Z, Qiao M, Li J, Li S, Xiang F. ARR12 promotes de novo shoot regeneration in Arabidopsis thaliana via activation of WUSCHEL expression. J Integr Plant Biol. 2017;59:747–58. Available from: http://www.ncbi.nlm.nih.gov/pubmed/28681564

  22. Meng WJ, Cheng ZJ, Sang YL, Zhang MM, Rong XF, Wang ZW et al. Type-B ARABIDOPSIS RESPONSE REGULATORs specify the shoot stem cell niche by dual regulation of WUSCHEL. Plant Cell. 2017;29:1357–72. Available from: http://www.ncbi.nlm.nih.gov/pubmed/28576846

  23. Kareem A, Durgaprasad K, Sugimoto K, Du Y, Pulianmackal AJ, Trivedi ZB et al. PLETHORA genes control regeneration by a two-step mechanism. Curr Biol. 2015;25:1017–30. Available from: http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=PMC4829346

  24. Varapparambath V, Mathew MM, Shanmukhan AP, Radhakrishnan D, Kareem A, Verma S et al. Mechanical conflict caused by a cell-wall-loosening enzyme activates de novo shoot regeneration. Dev Cell. 2022;57:2063–2080.e10. Available from: https://linkinghub.elsevier.com/retrieve/pii/S1534580722005482

  25. Lu X, Zhang Q, Wang Z, Cheng X, Yan H, Cai S et al. Development of an inducible DNA barcoding system to understand lineage changes in Arabidopsis regeneration. Dev Cell. 2024;1–15. Available from: https://doiorg.publicaciones.saludcastillayleon.es/10.1016/j.devcel.2024.10.023

  26. Metzger D, Clifford J, Chiba H, Chambon P. Conditional site-specific recombination in mammalian cells using a ligand-dependent chimeric Cre recombinase. Proc Natl Acad Sci U S A. 1995;92:6991–5.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  27. Brocard J, Feil R, Chambon P, Metzger D. A chimeric Cre recombinase inducible by synthetic,but not by natural ligands of the glucocorticoid receptor. Nucleic Acids Res. 1998;26:4086–90. Available from: https://academic.oup.com/nar/article-lookup/doi/https://doiorg.publicaciones.saludcastillayleon.es/10.1093/nar/26.17.4086

  28. Smetana O, Mäkilä R, Lyu M, Amiryousefi A, Sánchez Rodríguez F, Wu M-F et al. High levels of auxin signalling define the stem-cell organizer of the vascular cambium. Nature. 2019;565:485–9. Available from: http://www.nature.com/articles/s41586-018-0837-0

  29. Zhai N, Xu L. CRE/LOX-based analysis of cell lineage during root formation and regeneration in Arabidopsis. aBIOTECH. 2020;1:153–6. Available from: http://link.springer.com/https://doiorg.publicaciones.saludcastillayleon.es/10.1007/s42994-020-00025-y

  30. Wachsman G, Heidstra R, Scheres B. Distinct cell-autonomous functions of RETINOBLASTOMA-RELATED in Arabidopsis stem cells revealed by the brother of brainbow clonal analysis system. Plant Cell. 2011;23:2581–91. Available from: https://academic.oup.com/plcell/article/23/7/2581-2591/6097183

  31. Efroni I, Ip P-L, Nawy T, Mello A, Birnbaum KD. Quantification of cell identity from single-cell gene expression profiles. Genome Biol. 2015;16:9. Available from: https://biomedcentral-genomebiology.publicaciones.saludcastillayleon.es/articles/https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s13059-015-0580-x

  32. Efroni I, Mello A, Nawy T, Ip P-L, Rahni R, DelRose N et al. Root regeneration triggers an embryo-like sequence guided by hormonal interactions. Cell. 2016;165:1721–33. Available from: https://doiorg.publicaciones.saludcastillayleon.es/10.1016/j.cell.2016.04.046

  33. He Y, Zhang T, Sun H, Zhan H, Zhao Y. A reporter for noninvasively monitoring gene expression and plant transformation. Hortic Res. 2020;7:152. Available from: https://doiorg.publicaciones.saludcastillayleon.es/10.1038/s41438-020-00390-1

  34. Grefen C, Donald N, Hashimoto K, Kudla J, Schumacher K, Blatt MR. A ubiquitin-10 promoter-based vector set for fluorescent protein tagging facilitates temporal stability and native protein distribution in transient and stable expression studies. Plant J. 2010;64:355–65. Available from: https://onlinelibrary.wiley.com/doi/https://doiorg.publicaciones.saludcastillayleon.es/10.1111/j.1365-313X.2010.04322.x

  35. Tanaka A, Mita S, Ohta S, Kyozuka J, Shimamoto K, Nakamura K. Enhancement of foreign gene expression by a dicot intron in rice but not in tobacco is correlated with an increased level of mRNA and an efficient splicing of the intron. Nucleic Acids Res. 1990;18:6767–70. Available from: https://academic.oup.com/nar/article-lookup/doi/https://doiorg.publicaciones.saludcastillayleon.es/10.1093/nar/18.23.6767

  36. Nagaya S, Kawamura K, Shinmyo A, Kato K. The HSP terminator of Arabidopsis thaliana increases gene expression in plant cells. Plant Cell Physiol. 2010;51:328–32. Available from: https://academic.oup.com/pcp/article-lookup/doi/https://doiorg.publicaciones.saludcastillayleon.es/10.1093/pcp/pcp188

  37. He C, Chen X, Huang H, Xu L. Reprogramming of H3K27me3 is critical for acquisition of pluripotency from cultured Arabidopsis tissues. PLoS Genet. 2012;8:e1002911.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  38. Aoyama T, Chua N-H. A glucocorticoid-mediated transcriptional induction system in transgenic plants. Plant J. 1997;11:605–12. Available from: http://doi.wiley.com/https://doiorg.publicaciones.saludcastillayleon.es/10.1046/j.1365-313X.1997.11030605.x

  39. Clowes FAL. The immediate response of the quiescent centre to X-rays. New Phytol. 1970;69:1–18. Available from: https://nph.onlinelibrary.wiley.com/doi/https://doiorg.publicaciones.saludcastillayleon.es/10.1111/j.1469-8137.1970.tb04044.x

  40. Clowes FAL. Effects of β-radiation on meristems. Exp Cell Res. 1961;25:529–34. Available from: https://linkinghub.elsevier.com/retrieve/pii/0014482761901884

  41. Clowes FAL, Hall EJ. The quiescent centre in root meristems of Vicia faba and its behaviour after acute x-irradiation and chronic gamma irradiation. Radiat Bot. 1963;3:45–53. Available from: https://linkinghub.elsevier.com/retrieve/pii/0033756063900073

  42. Clowes FAL. Reorganization of root apices after irradiation. Ann Bot. 1959;23:205–10. Available from: https://academic.oup.com/aob/article/118718/Reorganization

  43. Matosevich R, Efroni I. The quiescent center and root regeneration. J Exp Bot. 2021; Available from: https://academic.oup.com/jxb/advance-article/doi/https://doiorg.publicaciones.saludcastillayleon.es/10.1093/jxb/erab319/6330663

  44. Subban P, Kutsher Y, Evenor D, Belausov E, Zemach H, Faigenboim A et al. Shoot regeneration is not a single cell event. Plants. 2020;10:58. Available from: https://www.mdpi.com/2223-7747/10/1/58

Download references

Acknowledgements

We thank Yubing He and Xiaozhao Xu for providing the RUBY template. We thank Liwen Bianji for editing the English text of a draft of this manuscript.

Funding

This work was supported by grants from the National Key R&D Program of China (2023YFE0101100/2024YFF1000700), the Strategic Priority Research Program of Chinese Academy of Sciences (Grant No. XDB0630000), the National Natural Science Foundation of China (32400281/32300285/32225007), Key R&D Program of Shandong Province, China (2024LZGC025), Key project at central government level: The ability establishment of sustainable use for valuable Chinese medicine resources (2060302-2401-08), China Postdoctoral Science Foundation (Grant No. 2023M733490), and Science and Technology Commission of Shanghai Municipality (24N12800100).

Author information

Author notes

  1. Shengnan Wang, Yu Li and Zijuan Li contributed equally to this work.

Authors and Affiliations

  1. School of Life Sciences, East China Normal University, Shanghai, 200241, China

    Shengnan Wang & Chao Li

  2. Key Laboratory of Plant Carbon Capture, CAS Center for Excellence in Molecular Plant Sciences, Shanghai Institute of Plant Physiology and Ecology, Chinese Academy of Sciences, Shanghai, 200032, China

    Yu Li, Zijuan Li, Lin Xu & Ning Zhai

  3. University of Chinese Academy of Sciences, Beijing, 100049, China

    Yu Li

Authors
  1. Shengnan Wang
    View author publications

    You can also search for this author inPubMed Google Scholar

  2. Yu Li
    View author publications

    You can also search for this author inPubMed Google Scholar

  3. Zijuan Li
    View author publications

    You can also search for this author inPubMed Google Scholar

  4. Chao Li
    View author publications

    You can also search for this author inPubMed Google Scholar

  5. Lin Xu
    View author publications

    You can also search for this author inPubMed Google Scholar

  6. Ning Zhai
    View author publications

    You can also search for this author inPubMed Google Scholar

Contributions

C.L., L.X., N.Z. conceived the study. S.W., Y.L., Z.L., and N.Z. performed experiments. L.X. and N.Z. wrote the manuscript. All authors discussed and analyzed the data.

Corresponding authors

Correspondence to Chao Li, Lin Xu or Ning Zhai.

Ethics declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

All authors agree with the submission of this manuscript to Plant Methods.

Competing interests

The authors declare no competing interests.

Additional information

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Wang, S., Li, Y., Li, Z. et al. Tracing meristem cell lineages during plant development and regeneration by the all-in-one CRE/LOX system. Plant Methods 21, 36 (2025). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s13007-025-01352-1

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s13007-025-01352-1

Keywords

Plant Methods

ISSN: 1746-4811